Background: Despite significant reductions in the morbidity and mortality associated with esophagectomy, survival for patients with esophageal cancer remains dismal. In most patients, adjunctive therapies including chemotherapy and radiation are beneficial. In fact, patients who achieve a complete pathologic response (pCR) after treatment with chemotherapy and radiation followed by esophagectomy often experience greatly improved survival. Unfortunately, the percentage of patients who currently achieve a pCR is low. Improving survival for esophageal cancer patients following esophagectomy requires that we ascertain how to increase the number of patients who achieve a pCR. In other malignancies, microRNAs (miRs) have been found to serve as effective diagnostic, prognostic, and predictive biomarkers. In addition, based on their ability to function as oncogenes or tumor suppressors, miR-based therapeutic approaches are currently being investigated. The role of miRs in the pathogenesis and treatment of esophageal cancer has not been thoroughly evaluated. Objectives: The primary objective of this study is to identify new biomarkers and therapeutic targets based an analysis of miR expression and function in esophageal cancer cells. Preliminary Findings: Our preliminary studies indicate that a) miR-214-3p and miR-199a-5p are dramatically downregulated in esophageal cancer cell lines compared to esophageal epithelial cells, b) this expression pattern has been verified in initial human tumor samples, c) specific binding interactions have been identified between miR-214-3p and miR-199a-5p with the oncogenic targets survivin and mitogen activated protein kinase kinase kinase 11 (MAP3K11), respectively, d) ectopic expression of miR-214-3p and miR-199a-5p results in a marked decrease in the levels of survivin and MAP3K11 in esophageal cancer cells, and e) ectopic expression of miR-214-3p and miR-199a-5p results in important functional consequences; specifically, increased sensitivity to chemotherapy-induced apoptosis and decreased proliferation, respectively. Based on these exciting observations, we HYPOTHESIZE that downregulation of miR-214-3p and miR-199a-5p occurs frequently in esophageal cancer cells and can be exploited for prognostic, predictive, and therapeutic purposes. Methods: To test this hypothesis, we propose 3 specific aims. (1) To characterize expression of miR- 214-3p and miR-199a-5p in human esophageal cancer specimens and correlate expression with clinical outcomes. Pretreatment biopsies of both tumor and normal esophageal epithelium will be obtained from 50 patients for evaluation of miR-214-3p and miR-199a-5p expression and correlation with outcomes. (2) To identify novel targets of miR-214-3p and miR-199a-5p that contribute to the development and progression of esophageal cancer. We will utilize human esophageal cancer cell lines to identify new targets and functions of miR-214-3p and miR-199a-5p in esophageal cancer cells. (3) To determine the anti-tumor efficacy of miR-214-3p and miR-199a-5p overexpression in vivo. Human esophageal cancer cell lines will be engineered to stably express selected miR-214-3p or miR-199a-5p. These engineered cells will then be implanted in nude mice. Their growth characteristics and response to chemotherapy will be compared to wild-type cells. Status: This is a resubmission. Impact: The incidence of esophageal cancer continues to increase in the Untied Sates, especially in males over the age of 50. Based on these demographics, this disease represents a significant problem in the VA healthcare system. Improving outcomes for esophageal cancer patients will greatly enhance the ability of the VA to fulfill its healthcare mission.